PROJECT SUMMARY/ABSTRACT Cholera is an acute diarrheal disease that affects an estimated to 3-5 million people each year. Cholera is caused by Vibrio cholerae; a gram-negative facultative human pathogen that is native to aquatic ecosystems around the world. The environmental distribution of V. cholerae makes it a health threat whenever populations lack access to good sanitation. The devastating consequences of cholera combined with the rapidity with which it can spread, evolving antibiotic resistance, and its ability to persist in the aquatic ecosystem, have underscored the need for the development of new approaches to limit the spread of this epidemic disease. V. cholerae ToxR is a membrane associated transcription factor that contains a periplasmic signaling domain tethered to a cytoplasmic DNA binding domain. ToxR has been shown to activate V. cholerae virulence factor production following entry into the host. Recent in our laboratory have suggested that ToxR may also function as a growth dependent virulence repressor. We showed that the accumulation of cell metabolites at high cell density could alter the activity of ToxR at its target promoters. Further, this process was dependent on the ToxR periplasmic signaling domain. These findings have extended the role of ToxR in V. cholerae pathogenesis and suggested the possibility that ToxR may contribute to a host-escape program that occurs late during infection. This proposal will build upon our findings to elucidate both the metabolic cues that drive the ToxR transition to a virulence repressor, and to identify the ToxR transcriptome and regulatory targets at high cell density. Two aims are proposed. In Aim 1 we will identify the direct and indirect targets of ToxR at high cell density. In Aim 2 we will identify V. cholerae genes that affect the activation state of ToxR. Identification of the regulatory target of ToxR and the genes that influence ToxR activity will illuminate important aspects of V. cholerae pathogenesis and provide a better understanding of the factors that contribute to the epidemic spread of this organism. Elucidating the mechanisms behind this phenotypic switch may contribute to the development of novel therapeutic approaches for the disease cholera.